Techniques for producing ethanol from lignocellulosic biomass, which does not compete with food, have been extensively developed. Lignocellulosic biomass may be degraded typically by treatment with saccharifying enzymes (e.g., cellulase) for fermentation by yeast.
The main components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Cellulose in the biomass has a rigid crystalline structure. In order to enzymatically degrade the lignocellulosic biomass, it is always necessary to pretreat the biomass to be easily in contact with and reacted with the enzyme.
As the pretreatment for the lignocellulosic biomass, methods using various principles have been proposed, which are genetically classified into physical treatment, chemical treatment, and biological treatment.
Examples of the physical treatment include finely pulverizing treatment. The finely pulverizing treatment is a method for finely pulverizing lignocellulosic biomass using a mill or the like. Enzymatic degradation (saccharification) is enhanced by the finely pulverizing treatment, for example, because increased contact area of biomass with enzymes by reduced particle size of biomass, decreased crystallinity and polymerization for cellulose, and the separation of cellulose molecules in units of microfibrils (Non-Patent Document 1).
Of these, methods for causing a chemical reaction (cleavage or formation of bonds) using a mechanical energy such as pulverization are referred to as mechanochemical treatment. As a mill for high impact and energy conservation, a tandem-ring mill using the ring-type pulverizing medium tumbling method has been developed (Non-Patent Document 2). When cedarwood that was preliminarily pulverized using a ball mill and then classified into a particle size of 200 μm or less with a sieve is subjected to the tandem-ring mill, the average particle size of the cedarwood reaches about 20 μm in 10 to 20 minutes, and when the pulverization is continued up to 60 minutes, the saccharification rate can be maximum. An example has been reported in which finely pulverized cedarwood obtained in the tandem-ring mill is saccharified with a commercially available cellulase preparation and is then fermented by yeast, so that ethanol is produced (Non-Patent Document 3).
Meanwhile, it has been shown that application of techniques for expressing various enzymes involved in saccharification of lignocellulose on the cell surface of microorganisms realizes a high ethanol yield from lignocellulosic biomass even with a relatively small amount of enzyme added. Non-Patent Document 4 has reported that, in ethanol production from hydrothermally treated rice straw, recombinant yeast Saccharomyces cerevisiae expressing three types of enzymes consisting of endoglucanase, cellobiohydrolase, and β-glucosidase on the cell surface degrades a cellulose moiety that fail to be degraded with a commercially available cellulase preparation alone, thereby exhibiting an ethanol yield higher than that of wild-type yeast.
However, in order to produce ethanol from lignocellulosic biomass, it is an important issue to reduce the use amount of enzyme necessary for saccharification, while maintaining or improving the saccharification efficiency of the enzyme. Even in the case where finely pulverized lignocellulosic biomass is used, if the use amount of enzyme is small, the saccharification and the fermentation yields may be significantly poor, and the process of recovering the enzyme from slurry after the fermentation may be complicated.
Furthermore, in order to increase the ethanol yield, it is necessary to increase the cellulase activity on the cell surface by expressing many types of enzymes at a high level. Furthermore, in order to allow the cellulase activity of the yeast on the cell surface to significantly act on increase in ethanol yield, a very high initial cell concentration is necessary. Such a high cell concentration leads to an increase in the ethanol production cost.